macrophages promote fibroblast growth factor receptor...

Angiogenesis, Metastasis, and the Cellular Microenvironment

Macrophages Promote Fibroblast Growth Factor Receptor-Driven Tumor Cell Migration and Invasion in aCxcr2-Dependent Manner

Laura R. Bohrer and Kathryn L. Schwertfeger

AbstractInfiltration of immune cells, specifically macrophages, into the tumor microenvironment has been linked to

increased mammary tumor formation and progression. Activation of growth factor receptor signaling pathwayswithin mammary epithelial cells, such as the fibroblast growth factor receptor 1 (FGFR1) pathway, inducesrecruitment of macrophages to the mammary epithelium. These macrophages promote increased epithelial cellproliferation and angiogenesis. However, the specific mechanisms by which these macrophages are regulated by thepreneoplastic epithelial cells and the mechanisms of action of the macrophages within the developing FGFR1-driven tumor microenvironment remain unknown. In this study, we show that activation of inducible FGFR1 inmammary glands leads to decreased activity of the TGFb/Smad3 pathway in macrophages associated with earlystage lesions. Further studies show that macrophages have increased expression of inflammatory chemokines thatbind Cxcr2 following exposure to conditioned media from mammary epithelial and tumor cells in which the FGFpathway had been activated. The increase in these ligands is inhibited following activation of the TGFb pathway,suggesting that decreased TGFb signaling contributes to the upregulation of these chemokines. Using coculturestudies, we further show that macrophages are capable of promoting epithelial and tumor cell migration andinvasion through activation of Cxcr2. These results indicate that macrophage-derived Cxcr2 ligands may beimportant for promoting mammary tumor formation regulated by FGFR signaling. Furthermore, these resultssuggest that targeting Cxcr2 may represent a novel therapeutic strategy for breast cancers that are associated withhigh levels of infiltrating macrophages. Mol Cancer Res; 1–12. �2012 AACR.

IntroductionBreast tumor formation and progression involve complex

interactions between tumor cells and their surroundingenvironment. Infiltration of immune cells into the tumormicroenvironment has been linked to tumor formation andprogression (1). Specifically, increased numbers of tumor-associated macrophages are linked to poor prognosis inbreast cancer patients (2). Although macrophages wereinitially expected to inhibit tumor growth via their cytotoxicfunctions, it is now clear that exposure to the tumormicroenvironment polarizes macrophages towards atumor-promoting phenotype (3). Therefore, obtaining abetter understanding of the mechanisms through which

macrophages regulate tumor growth and progression mayresult in the development of strategies that either inhibit theactivities of tumor-promoting macrophages or reprogramthe macrophages to increase their tumor cytotoxic activities.Numerous functions have been ascribed to macrophagesduring tumor progression, including promotion of tumorcell invasion, angiogenesis and immune suppression (4).Although published studies have identified specific mechan-isms through which macrophages contribute to mammarytumor metastasis in mouse models (5, 6), less is knownregarding the mechanisms of macrophage function duringmammary tumor initiation.Macrophages associated with late stage tumors have been

shown to express high levels of TGFb (7). TGFb, which is akey regulator of development, immune function and woundhealing, acts primarily through a core-signaling pathwayinvolving the Smad family of transcription factors (8).During mammary gland development, TGFb is a potentinhibitor of epithelial cell proliferation and branching mor-phogenesis (9). In premalignant lesions, TGFb acts as atumor suppressor by inhibiting proliferation and promotingapoptosis. Paradoxically, malignant tumors are associatedwith high levels of TGFb, which promote later stages oftumor progression and metastasis (8, 10). Pleiotropic effectsof TGFb have also been observed in macrophages,

Authors' Affiliation: Department of Laboratory Medicine and Pathologyand Masonic Cancer Center, University of Minnesota, Minneapolis,Minnesota

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Kathryn L. Schwertfeger, Department of Labora-tory Medicine and Pathology and Masonic Cancer Center, University ofMinnesota, 420 Delaware St. SE, Minneapolis, MN 55455, USA. Phone:612-626-9419; Fax: 612-626-2600; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-12-0275

�2012 American Association for Cancer Research.

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depending on their stage of differentiation. Although TGFbacts as a chemoattractant for monocytes, it inhibits phago-cytosis and production of proinflammatory mediators indifferentiated macrophages (11). TGFb expression levels arehigh in macrophages associated with late stage tumors (7),which is thought to influence many aspects of malignantprogression including invasion, angiogenesis and immunesuppression (10). However, the functional consequences ofregulating the TGFb/Smad signaling pathway withinmacrophages during different stages of tumorigenesis havenot been investigated.Inappropriate activation of growth factor signaling path-

ways has been strongly linked to breast cancer formation andprogression. Recent studies have implicated the fibroblastgrowth factor (FGF) pathway in tumor growth, progressionand resistance to standard therapies (12, 13). Amplificationof the chromosomal region of 8p12 that includes the FGFreceptor 1 (FGFR1) gene is associated with poor prognosis,and approximately 10% of breast cancers exhibit amplifiedFGFR1 (13, 14). Transgenic mice expressing an inducibleFGFR1 (iFGFR1) transgene in mammary epithelial cellsdevelop early epithelial lesions that progress to alveolarhyperplasia, ultimately resulting in mammary tumor forma-tion (15). Activation of iFGFR1 leads to alterations in themicroenvironment, including increased angiogenesis and arapid inflammatory response characterized by infiltratingmacrophages (15, 16). Macrophage depletion in this modelleads to reduced epithelial cell proliferation and angiogenesisassociated with early stage lesions showing that in anFGFR1-dependent model of mammary tumor formation,macrophages are capable of promoting the development ofearly stage epithelial lesions (16).In these studies, we have further used the iFGFR1 model

to identifymechanisms that regulate the protumor functionsof macrophages during early stage tumor formation. Weshow here that macrophages associated with iFGFR1-drivenearly stage epithelial lesions exhibit decreased activation ofthe TGFb/Smad3 pathway. The decrease in TGFb-associ-ated genes within macrophages correlates with increasedexpression of macrophage-derived chemokines that bind tothe chemokine receptor Cxcr2. Restoration of TGFb sig-naling leads to inhibition of expression of these chemokinesin macrophages. These studies suggest that repressed TGFb/Smad3 signaling may be functionally important for regu-lating the protumorigenic function of macrophages in earlystages of tumor formation. Furthermore, these studies showthat macrophage-derived chemokines, specifically Cxcr2binding chemokines, promote migration and invasion ofpreneoplastic mammary epithelial cells, suggesting a poten-tial therapeutic target for early stage breast tumors.

Materials and MethodsAnimalsGeneration of mouse mammary tumor virus (MMTV)-

iFGFR1 transgenic mice has been described previously (15)and the mice were obtained from Dr. Jeffrey Rosen (BaylorCollege of Medicine, Houston, TX). Animal care and

procedures were approved by the Institutional Animal Careand Use Committee of the University of Minnesota andwere in accordancewith the procedures detailed in theGuidefor Care and Use of Laboratory Animals.

Cell sorting and RT-PCR analysisSix-week-old female MMTV-iFGFR1 transgenic mice

and nontransgenic littermates were injected intraperitone-ally (i.p.) with 1mg/kg B/B dimerizer (Clontech).Mice weresacrificed 48 hours later andmammary glands were collectedfor analysis. The tissue was dissociated using 2 mg/mLcollagenase A (Roche Applied Science) for 45 minutes at37�C with rocking at 200 rpm. The solutions were vigor-ously shaken every 15 minutes and the dissociated cells werecollected by centrifuging for 5 minutes at 1,500 rpm. Thecells were washed 3 times with DMEM/F12 containing 5%FBS at 1,500 rpm and 2 times at 800 rpm for 5minutes each.The cells were stained with either Cd11b-APC (Life Tech-nologies) at a dilution of 1:200 or isotype control antibodyat the same concentration for 1 hour at RT. The cellswere then washed, filtered through a 40 mm filter and sortedusing a triple laser MoFlo (Cytomation). RNA was isolatedfrom Cd11b-positive cells sorted from 6 mice per timepointas described above and pooled into duplicate samples. RNAwas extracted using the Arcturus PicoPure RNA IsolationKit (Life Technologies) and RT-PCR analysis was carriedout using primers specific for ArgI and iNOS as describedbelow. qRT-PCR analysis was carried out for TGFb1 asdescribed below. Primer sequences are listed in Supplemen-tary Table 1. Cyclophilin was used to normalize geneexpression levels.

ImmunofluorescenceMammary glands fromMMTV-iFGFR1 transgenicmice,

or nontransgenic littermate controls, that were treated withB/B for 48 hours as described above were fixed for 2 hours in4% paraformaldehyde and embedded in paraffin. Fivemicrometers sections were used for immunofluorescentstaining using the following antibodies and dilutions: ratmonoclonal F4/80 (Invitrogen), 1:50; phospho-Smad3(Cell Signaling), 1:200. Immunostaining was carried outas described previously (17) in the absence of antigenretrieval. F4/80 and phospho-Smad3 positive cells werecounted and double positive cells were calculated relativeto the total number of F4/80 positive cells. A minimum of600 cells from a total of 3 mice per treatment group wascounted for each dataset. All statistical analyses were carriedout using the unpaired Student t test to compare 2 means.

Cell cultureHC-11 cells were maintained in RPMI containing 10%

FBS (Invitrogen), 1% penicillin-streptomycin, 5 mg/mLinsulin (Sigma-Aldrich) and 10 ng/mL EGF (Invitrogen).HC-11 cells stably expressing the iFGFR construct (HC-11/R1) were generated as previously described (18) and wereacquired from Dr. Jeffrey Rosen. HC-11/R1 cells weremaintained in HC-11 medium with the addition of 0.7mg/mL puromycin (Sigma-Aldrich). We received SUM225

Bohrer and Schwertfeger

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andMCF10DCIS.com fromDr. Fariba Behbod (Universityof Kansas City Medical Center, Kansas City, KS) andmaintained as described (19). All other cell lines wereobtained from the American Type Culture Collection(ATCC) and maintained in the suggested media. All celllines were used for fewer than 6 months after resuscitation.HC-11 cells and HC-11/R1 cells, which are not commer-cially available, are not maintained for longer than 20passages and are tested for mycoplasma, b-casein expressionand iFGFR1 expression regularly.

Two-dimensional coculture assaysHC-11/R1 cells were grown to confluence, washed with

PBS and incubated overnight in serum-free RPMI. The cellswere treated with 30 nmol/L B/B or an equal amount ofethanol as solvent control for 24 hours. Conditioned mediawas collected (R1-CM) and added to RAW 264.7 cells thathad been incubated overnight in serum-free DMEM. Todetermine gene expression, cells were collected in Trizol after2 hours of treatment with R1-CM supplemented with orwithout 10 ng/mL recombinant TGFb (R&D Systems,Minneapolis, MN, USA) and analyzed as described below.For signaling studies, R1-CMwas added to RAW264.7 cellsfor the indicated time and lysates were collected for immu-noblotting. Then, RAW 264.7 cells were pretreated for 30minutes with 2.5 or 10 mmol/L of the MEK1 inhibitorU0126 (Cell Signaling) or solvent control (DMSO) beforeincubation with R1-CM and the inhibitor or control for anadditional 2 hours. For migration assays, RAW 264.7 cellswere incubated for 24 hours with R1-CM and CM wascollected (R1/RAW-CM).Migration assays were carried outusing cell culture inserts with 8 mm pore size (BD Bios-ciences, San Jose, CA, USA). HC-11 cells were washed withPBS and incubated overnight in serum-free RPMI. Cellswere plated on the top of the insert and either R1-CMor R1/RAW-CM was placed on the bottom of the insert as achemoattractant. The CXCR2 inhibitor, SB225002 (20 or200 nmol/L, Cayman Chemical), or the solvent control,ethanol, was added with the HC-11 cells to the top of thewell. After 18 hours, cells on the bottom of the insert werefixed with 4% paraformaldehyde and stained with hema-toxylin. The number of cells migrated in 4 fields of the insertwere counted. Similar experiments were done with humancell lines in which MCF-7 cells were treated with 50 ng/mLbasic fibroblast growth factor (bFGF, Invitrogen). After 24hours, the conditioned media (MCF-CM) was then incu-bated with the human monocyte cell line THP-1 that hadbeen treated with 5 ng/mL PMA 24 hours and then starvedovernight. Gene expression of CXCR2 ligands in macro-phages was determined by collecting THP-1 cells treatedwith MCF-CM for 2 or 4 hours and analyzed as describedbelow. Formigration assays,MCF-CMwas added toTHP-1cells for 24 hours and the conditioned media (MCF/THP-CM) was used as a chemoattractant for MCF-7 cells in thecell culture inserts as described above. The CXCR2 inhib-itor, SB225002 (100, 200, or 400 nmol/L) or solventcontrol, ethanol, was added with MCF-7 cells in the topof the well.

Quantitative reverse transcription-PCRRNA was extracted from primary macrophages or mac-

rophage cell lines (RAW 264.7 and THP-1, ATCC) usingTrizol (Invitrogen) as described in the manufacturer proto-col. cDNA was prepared using the Quantitect reversetranscription kit (Qiagen). Quantitative RT-PCR (qRT-PCR) was carried out using SYBR green (Bio-Rad) and theBio-Rad iQ5 system. The 2�DDCt method (20) was used todetermine the relative quantification of gene expressionnormalized to cyclophilin. The primers used for these studiesare listed in Supplementary Table 1.

qRT-PCR arrayRNAwas isolated from RAW264.7 cells treated with R1-

CM for 2 hours in the presence or absence of 10 ng/mLTGFb, and cDNA was made, as described above. The RT2

Profiler PCR Array: Mouse chemokines and receptors fromSA Biosciences was carried out as described for other qRT-PCR experiments.

ELISA analysis and activity assaysR1-CM was added to starved RAW 264.7 cells in the

presence or absence of 10 ng/mL TGFb. After 2 hoursthe media was removed and serum free RPMI was added.The R1/RAW-CM was collected after an additional 24hours and ELISAs were carried out to quantify the level ofCxcl1, Cxcl5, IL-10, and IL-12 following themanufacturer'sprotocol (R&D Systems). In addition, the RAW 264.7 werecollected in lysis buffer (10 mmol/L Tris-HCl [pH 7.4]containing 1 mmol/L pepstatin A, 1 mmol/L leupeptin, and0.4% Triton X-100) and the supernatant was used for anarginase activity assay (BioAssay Systems). For the Smad3luciferase assay, RAW 264.7 cells were transduced with alentivirus expressing a Smad3 reporter construct (Qiagen)and were then treated with conditioned media from B/B-treated HC-11/R1 cells for 6 hours. Luciferase activity wasmeasured using standard procedures (Promega). For analysisof human chemokine expression, THP-1 cells were incu-bated with MCF-CM for 2 hours and then serum freeDMEM for 24 hours. The levels of secreted CXCL1 andCXCL8 in the MCF/THP-CM were measured by ELISAfollowing the manufacturer's protocol (R&D Systems).

Immunoblot analysisCells were lysed in RIPA buffer and equal amounts of

protein were analyzed by SDS-PAGE. Immunoblotting wasdone using the following antibodies: CXCR2 (AHR1532Z;Biosource), b-tubulin (2146; Cell Signaling), pERK1/2(9101; Cell Signaling), and ERK1/2 (sc-94; Santa CruzBiotechnology).

Three-dimensional coculture assayMammary glands were isolated from 8- to 12-week-old

MMTV-iFGFR1 transgenic mice as described above. Bonemarrow was collected from femurs of 6- to 10-week-oldwild-type (WT) mice. Cells were differentiated into macro-phages with the addition of 20% conditioned media fromL929 cells that have a high concentration of granulocyte

Cxcr2 Contributes to Macrophage-Induced Tumor Cell Migration

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colony-stimulating factor (G-CSF). A total of 10,000 mam-mary epithelial cells (MECs) were plated on growth-factorreduced matrigel (BD Biosciences) in 8 well chamber slidesas described previously (21). After 2 days, 3000 bonemarrow derived macrophages (BMDM) were added to the3D culture. Also, the following treatments began at thistime: �30 nmol/L B/B and �200 nmol/L SB225002,CXCR2 inhibitor (with (�) being ethanol, solvent control).After 10 days, cells were fixed with 2% paraformaldehydeand costained for macrophages (F4/80, Invitrogen) andepithelial cells (cytokeratin 8 (ab59400- Abcam) andmounted with ProLong Gold antifade reagent with DAPI(Invitrogen). Images were taken at the confocal microscopyfacility at the Masonic Cancer Center (University of Min-nesota, Minneapolis, MN).

ResultsRegulation of pro- and anti-inflammatory genes inmacrophages following exposure to conditioned mediafrom iFGFR1-activated HC-11/R1 cellsAs described previously, activation of iFGFR1 in mam-

mary epithelial cells promotes rapid recruitment of macro-phages to epithelial structures in vivo and in vitro (16).Furthermore, depletion of macrophages leads to delayed

formation of early hyperplastic lesions in vivo (16). There-fore, we further used this model to delineate themechanismsthat mediate the interactions between epithelial cells andmacrophages. We have previously shown that exposure ofmacrophages to conditioned media from HC-11/R1 cellsfollowing activation of iFGFR1 leads to increased produc-tion of the proinflammatory cytokine IL-1b bymacrophagesin vitro and that IL-1b contributes to the formation of earlystage lesions in vivo (17). To extend our analysis of macro-phage response to iFGFR1 activation in epithelial cells, wefurther analyzed the expression of various genes known to bepreferentially regulated in tumor associated macrophages,including IL-10, IL-12, Arginase I (ArgI), and TGFb. Forthese studies, RAW 264.7 cells were exposed to conditionedmedia from HC-11/R1 cells treated with either the B/Bhomodimerizer, which activates the iFGFR1, or ethanol as asolvent control. After exposure to conditioned media,RAW 264.7 cells were analyzed for expression and activityof Arg1, IL-10, IL-12, and TGFb. As shown in Fig. 1,expression levels of IL-10 and gene expression and activity ofArgI, which are typically increased in tumor associatedmacrophages, were induced whereas expression levels ofIL-12, which are normally reduced in tumor associatedmacrophages, were decreased. Interestingly, expression of

Figure 1. Exposure to conditioned media from mammary epithelial cells with activated iFGFR1 leads to regulation of inflammation-associated genes in vitro.RAW 264.7 cells were exposed to conditioned media from B/B-treated HC-11/R1 cells for 2 hours and either cells were collected for gene expression orserum free media was added for 24 hours to examine secreted protein expression or activity. A, qRT-PCR analysis of ArgI gene expression levels(left panel) and arginase activity (right panel). B, qRT-PCR analysis of IL-10 gene expression levels (left panel) and secreted protein level by ELISA (right panel).C, qRT-PCR analysis of IL-12 gene expression levels (left panel) and secreted protein level by ELISA (right panel). D, qRT-PCR analysis of TGFb1gene expression levels (left panel) and Smad3 transcriptional activity by luciferase assay (right panel). Expression levels were normalized to cyclophilin forqRT-PCR. Error bars represent standard error of the mean (SEM). �, P < 0.05; ��, P < 0.005; ��, P < 0.001.






the TGFb1 gene, which is usually induced in tumor asso-ciated macrophages, was reduced in these studies. In addi-tion, the transcriptional activity of Smad3 also decreased(Fig. 1D) suggesting an overall decrease in the TGFb/Smad3pathway. These results, together with our previously pub-lished studies (17), show that exposure of macrophages toconditioned media from epithelial cells with activatediFGFR1 leads to regulation of a number of genes associatedwith macrophage polarization.

Decreased TGFb gene expression and Smad3 activity inmacrophages from MMTV-iFGFR1 transgenic miceTo examine gene expression of macrophages in vivo,

macrophages were isolated from the mammary glands ofWT and MMTV-iFGFR1 transgenic mice following 48hours of B/B treatment, at which time macrophage recruit-ment to epithelial structures is consistently observed (16).Following a brief enzymatic digestion, macrophages wereisolated by sorting with anti-Cd11b, which stains bothmonocytes and macrophages. Approximately 8.5% of the

cells isolated from the mammary glands were positive for theCd11b antigen compared with the isotype control antibody(Supplementary Fig. 1A). These cells were collected for bothimmunostaining and RNA extraction. To determine thepercentage of the population of sorted cells that representsmature macrophages, staining was carried out using the F4/80 antibody (Supplementary Fig. 1B). Approximately 85%of the sorted cells were positive for F4/80, suggesting that thepreparation was significantly enriched for macrophages. Toexamine gene expression in the macrophages, RT-PCRanalysis was carried out and expression levels of ArgI, amarker of protumor macrophages, and iNOS, a marker oftumor-inhibitory macrophages, were examined. As shownin Fig. 2A, ArgI expression was increased and iNOS expres-sion was decreased in macrophages isolated from MMTV-iFGFR1 transgenic mice treated with B/B, consistent with apolarization of the macrophages towards the tumor-promot-ing phenotype. Further studies using qRT-PCR revealed adecrease in expression of TGFb1 in these macrophages (Fig.2B), consistent with the results observed in vitro (Fig. 1).

Figure 2. Alterations in the TGFb gene expression and Smad3 activity in macrophages isolated frommammary glands following FGFR1 activation. A, Cd11b-positive sorted cells were analyzed for expression of the indicated genes using RT-PCR. The lanes represent macrophages sorted from 3 MMTV-iFGFR1transgenic (Tg) or 3 nontransgenic WT littermates and pooled. B, expression of TGFb1 in macrophages isolated from MMTV-iFGFR1 transgenicmice or nontransgenic WT littermates treated with B/B for 48 hours. Expression levels were normalized to cyclophilin. C, nontransgenic (i, ii) andtransgenic (iii, iv) mice were treated with B/B for 48 hours andmammary glands were collected. Sections were costained with the F4/80 and phospho-Smad3antibodies. F4/80 positive cells (red) are found in the stroma surrounding the epithelial structures (blue ¼ DAPI staining of nuclei). Phospho-Smad3(green) is detected in nuclei of F4/80 positive cells in sections from the nontransgenic (arrow, ii) but not transgenic (arrow, iv), mice. Scale bars ¼ 50 mm.D, quantification of the percentage of F4/80þ/phospho-Smad3þ cells. Error bars represent SEM. �, P < 0.05; ��, P < 0.0001.






To determine whether decreased TGFb productioncorrelated with altered Smad3 activity in the macrophages,mammary gland sections from WT and MMTV-iFGFR1transgenic mice following 48 hours of B/B treatment wereimmunostained with an antibody to phosphorylatedSmad3 (pSmad3; Fig. 2C). The sections were costainedwith an F4/80 antibody to visualize the macrophages andthe percentage of F4/80 cells positive for pSmad3 expres-sion was determined (Fig. 2D). In WT mice, approxi-mately 25% of the F4/80þ cells located within the stromasurrounding the ductal structures were positive forpSmad3. Interestingly, there was a significant decrease inthe number of cells positive for pSmad3 present in thestroma following 48 hours of B/B treatment of iFGFR1mice. This observation suggests that along with decreasedexpression of TGFb in the macrophages, there is alsodecreased TGFb signaling within macrophages followingiFGFR1 activation in mammary epithelial cells, consistentwith the in vitro studies.

Effects of TGFb on chemokine expression inmacrophagesOn the basis of our results, we hypothesized that decreased

TGFb signaling in macrophages may be linked to protu-morigenic macrophage function in early stages of tumori-genesis. Activation of the TGFb/Smad3 pathway has beenshown to inhibit the expression of inflammatory cytokinesand chemokines (22, 23), suggesting that repression of thispathway leads to induction of these factors. Furthermore,recent studies showed that loss of signaling through Tgfbr2led to increased expression of chemokines in a model ofpancreatic ductal adenocarcinoma (24). Therefore, we used aqRT-PCR-based chemokine array to analyze chemokinegene expression in macrophages exposed to conditionedmedia from B/B treated HC-11/R1 cells in the absence orpresence of exogenous TGFb. Several chemokines werefound to be either up- or downregulated in the macrophagesfollowing exposure to these conditions (Table 1). Thechemokines were divided into 5 clusters based on the changein their expression patterns. Although a number of geneswere either induced or repressed with B/B alone and with B/B and TGFb combined, we focused on genes in cluster 1,which were upregulated with B/B treatment and down-regulated with the addition of TGFb. Interestingly, 4 ofthese chemokines (Cxcl1, Cxcl2, Cxcl5, and Cxcl7) areligands for the Cxcr2 receptor. Before validating expressionlevels of these chemokines, we confirmed that Cxcr2 isexpressed in the HC-11 mammary epithelial cell line byimmunoblot analysis (Fig. 3A). We validated the resultsfrom the array using distinct primer sets to determine geneexpression and ELISAs to measure secreted proteins. Weobserved similar gene and protein expression patterns forboth Cxcl1 and Cxcl5 (Fig. 3B). Induction of Cxcl2 andCxcl7 was not detected by ELISA (data not shown). Thesedata suggest that macrophages respond to FGFR1-inducedsoluble factors by increasing the production of chemokinesthat bind Cxcr2, primarily Cxcl1, and Cxcl5, and that thisinduction can be inhibited by restoring TGFb signaling.

Next, we wanted to determine the signaling pathway bywhich the Cxcr2 ligands are regulated in the macrophages.Expression of Cxcr2 ligands can be regulated by varioussignaling pathways depending on cell type and stimulus,including the NFkB and ERK pathways (24, 25). Interest-ingly, we were unable to detect an increase in NFkB activityin macrophages exposed to conditioned media from B/B-treated HC-11/R1 cells (data not shown). Further analysisfocused on examining the ERK signaling pathway. For thesestudies, RAW 264.7 cells were exposed to conditionedmedia from HC-11/R1 cells treated with or without B/B.Protein lysates were collected at different time points and theexpression of phosphorylated and total ERK1/2 was exam-ined. There was a rapid induction of ERK1/2 activation inmacrophages treated with B/B conditioned media in com-parison to solvent controls (Fig. 3C). Further studies weredone to determine the contribution of the ERK1/2 pathwayto expression of the Cxcr2 ligands. As shown in Fig. 3D,blocking ERK activation with the MEK1 inhibitor U0126led to a decrease in gene expression of the Cxcr2 ligands.Inhibition of Cxcl1 was observed at the lowest concentrationof U0126, whereas there was a dose-dependent decrease inCxcl5 expression (Fig. 3D). These results suggest thatalthough both ligands are regulated by the ERK pathway,

Table 1. Macrophage chemokines regulated byconditioned media from FGFR1-inducedmammary epithelial cells in the presence orabsence of TGFb

Fold change Fold change

Gene ID B/B CM B/B CMþTGFb

Cluster 1CCL8 3.9 0.93CCR1 1.86 0.863CCR1/1 2.04 0.833CXCL1 1.44 0.81CXCL2 1.79 0.716CXCL5 3.25 1.85CXCL7 1.47 0.94

Cluster 2Bmp6 0.65 1.07CCL2 0.172 0.776CCL20 0.429 1.18Rgs3 0.425 2.02

Cluster 3CX3CL1 1.76 3.15Trem1 2.32 8.05

Cluster 4Ccbp2 0.39 0.3CCR8 0.54 0.53Ecgf1 0.42 0.55

Cluster 5CCR6 3.14 3.75TNfsf14 1.97 1.73Xcl1 1.82 2.02






they may be regulated via slightly different mechanisms.Overall, these results indicate that the ERK pathway con-tributes to the regulation of Cxcr2 ligand expression.

Macrophages induce migration of epithelial cells, whichis blocked by Cxcr2 inhibitionTo determine the effects of Cxcr2 ligand secretion from

macrophages, we developed a coculturemigration assay (Fig.4A). In this assay, conditionedmedium from theHC-11/R1cells containing FGFR1-induced soluble factors was addedto RAW 264.7 cells and allowed to incubate overnight. Theability of the soluble factors collected from the treated RAW264.7 cells to promote migration of parental HC-11 cells,which do not respond to B/B treatment, was determinedusing a transwell assay. The ability of macrophages topromote epithelial cell migration following exposure toconditioned media from cells with an activated FGFR1reflects the ability of the stimulated macrophages to act ina tumor-promoting manner. Conditioned media from B/Btreated HC-11/R1 cells alone was able to induce migrationof HC-11 cells, suggesting that activation of FGFR1 in thesecells leads to the production of migratory factors (Fig. 4B).

However, the conditioned media isolated from the treatedmacrophages led to a significant increase in migration ofHC-11 cells compared with conditioned media from theHC-11/R1 cells alone (Fig. 4B). Therefore, treatment ofmacrophages with conditioned media following FGFR1activation leads to an increase in the ability of macrophagesto produce migratory factors. To determine whether theincrease inmigration required Cxcr2, we further assessed theeffects of blocking Cxcr2 activity on HC-11 cell migrationusing the Cxcr2-specific inhibitor SB225002. As shownin Fig. 4B, the increase in migration was blocked whenSB225002 was incubated with the HC-11 cells in thepresence of the macrophage conditioned media. Theseresults show an important role for macrophage-derivedCxcr2 ligands in the promotion of mammary epithelial cellmigration.

Macrophages promote invasion of primary mammaryepithelial cells in a 3D coculture assayPrimarymammary epithelial cells (MEC) form acinar-like

structures when grown in 3D culture and more closelyrepresent characteristics of mammary epithelium in vivo

Figure 3. Activation of iFGFR1 in mammary epithelial cells leads to increased expression of Cxcr2-binding chemokines in macrophages. A, HC-11 cells weretreated with R1/RAW-CMwith or without B/B. Whole cell lysates were collected and the expression of Cxcr2 and b-tubulin (loading control) was analyzed byimmunoblotting. B, RAW 264.7 cells were treated with R1-CM in the presence or absence of 10 ng/ml TGFb for 2 hours. Expression of the givengeneswas determined by qRT-PCR and normalized to cyclophilin (left panel). For protein expression, RAW264.7 cells were incubatedwith R1-CM for 2 hoursfollowed by serum free media for 24 hours. The conditioned media samples were analyzed using ELISAs (middle and right panel). C, RAW 264.7cells were treated with R1-CM for the given time. Whole cell lysates were collected and the expression of pERK1/2 and ERK1/2 (loading control) wasanalyzed by immunoblotting. D, RAW 264.7 cells were pretreated with DMSO (solvent control) or U0126 for 30 minutes and then treated for anadditional 2 hours with the same conditions in the presence of B/B R1-CM. The expression of the given genes was analyzed by qRT-PCR and normalizedto cyclophilin. Error bars represent SEM. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.






(21). We took advantage of the ability of primary MECsisolated from MMTV-iFGFR1 transgenic mice to grow in3D culture (21) and developed a coculture model to studythe interactions betweenMECs and macrophages. For these

studies, we isolated MECs from iFGFR1 transgenic miceand plated them in growth factor reduced Matrigel. After 2days, bone marrow derived macrophages (BMDM) thatwere isolated from WT mice, were added to MECs. Thestructures were grown in the presence or absence of B/B andtheCXCR2 inhibitor SB225002. After 10 days of treatment,cells were fixed and stained for epithelial (cytokeratin 8) andmacrophage (F4/80) markers. As shown in Fig. 5A, additionof B/B led to larger MEC structures, consistent with pre-viously published studies (21). Although coculture ofMECs with BMDM did not significantly affect size ofthe structures (data not shown), a significant increase inthe number of invasive structures was observed (Fig. 5Aand B). With the addition of the CXCR2 inhibitor, macro-phages were still recruited to the MECs (Fig. 5A), but thepercentage of invasive structures was significantly inhibited(Fig. 5A and B). These data suggest that Cxcr2 ligandssecreted from primary macrophages induce invasion ofprimary MECs.

Human macrophages secrete CXCR2 ligands thatpromote migration of human breast cancer cellsTo verify the results of the mouse iFGFR1 system, we

used human breast cancer cell lines. We first examinedthe expression of CXCR2 in different breast cell lines:normal (MCF10A), pre-invasive (MCF10DCIS.com andSUM225), estrogen receptor positive (MCF-7 and T47D)and triple negative (MDA-MB-231, 435A, and 468)

Figure 4. Cxcr2 inhibition leads to decreased macrophage-inducedmigration of HC-11 cells. A, model of the migration coculture assay. B,R1-CM, �/þ B/B, was exposed to RAW 264.7 cells for 24 hours and theconditioned media, R1/RAW-CM was used as the chemoattractant for atranswell migration assay. HC-11 cells were plated on top of the insertwith ethanol (solvent control) or CXCR2 inhibitor (þ, 20 nmol/L and þþ,200 nmol/L). After 18 hours, themigrated cells on the bottom of the insertwere stained with hematoxylin and counted. Error bars represent SEM.��, P < 0.001.

Figure 5. Macrophages induceinvasion of primary mammaryepithelial cells grown in 3D culture.A, primaryMECs from iFGFR1micewere grown in Matrigel in theabsence or presence of BMDM.Every 2 to 3 days fresh media wasadded with the treatments: �30nmol/L B/B and �200 nmol/LCXCR2 inhibitor. After 10 days oftreatment, cells were fixed andimmunostained with cytokeratin 8(green) and F4/80 (red). DAPI (blue)labeled nuclei. Arrows indicateinvasive structures. Left imageslight microscopy. Scale bars ¼ 50mm. B, for each treatment,approximately 80 structures wereexamined for invasion from 3independent experiments. Errorbars represent SEM. �, P < 0.05;��, P < 0.01.






(Fig. 6A). As MCF-7 cells express CXCR2 and they havepreviously been shown to respond to FGF treatment (26),we chose to use these cells to determine the ability of bFGFstimulation to promote CXCR2 ligand induction in macro-phages. MCF-7 cells were treated with or without bFGF and

the MCF-7 conditioned media was added to the humanmonocyte cell line THP-1 that had been differentiated tomacrophages with PMA as described (27). After 2 and 4hours, THP-1 cells were collected for gene expressionanalysis. In addition to the Cxcr2 ligands in mice, humancells also express CXCL8. Gene expression of all CXCR2ligands, with the exception of CXCL7 (data not shown),increased following exposure to conditioned media fromMCF-7 cells treated with bFGF (Fig. 6B). Further analysis ofprotein expression showed significant increases in secretionof both CXCL1 and CXCL8 (Fig. 6B), whereas the otherligands were not expressed at detectable levels by ELISA(data not shown). bFGF treatment of THP-1 cells alone didnot induce expression of these chemokines, suggesting thatthese results are not due to the direct action of bFGF onmacrophages (data not shown). The MCF-7/THP-1 con-ditioned media was used as the chemoattractant in a trans-well migration assay to examine the migration of MCF-7cells. Media from MCF-7 cells exposed to bFGF increasedmigration compared to no treatment and there was anadditional increase when the media was also exposed toTHP-1 cells (Fig. 6C). Finally, migration was inhibited witha CXCR2 inhibitor (Fig. 6C), showing that the CXCR2ligands are important for macrophage-induced breast cancercell migration.

DiscussionRecent studies have showed that FGFR signaling con-

tributes to breast cancer growth and resistance to conven-tional therapies (12, 13). Our studies focus on understand-ing how activation of FGFR1 in tumor cells leads toprotumorigenic alterations in the tumor microenvironment.We describe here a novelmechanism of paracrine interactionbetween tumor cells and macrophages that is driven byFGFR1 activation in the tumor cells and chemokine expres-sion in the macrophages. We have previously showed thatactivation of iFGFR1 in the HC-11/R1 cells results in theinduction of a number of secreted factors that can influencemacrophage recruitment (16) and cytokine production (17).The studies described here further analyze the mechanismsthroughwhichmacrophages contribute to early stage tumor-igenesis. Although carrying out coculture studies, we found anumber of genes to be regulated in an expected mannerbased on published studies of gene expression in tumorassociated macrophages, including increased ArgI and IL-10and decreased IL-12 (28, 29). Interestingly, however,decreased expression of TGFb gene expression was consis-tently observed in the macrophages both in vivo and in vitro,which is not consistent with the phenotype of macrophagesassociated with late stage tumors (30). Although levels ofTGFb protein were not detectable by ELISA in the in vitrococulture model (data not shown), a decrease in Smad3activity was observed in the macrophages, consistent withthe decrease in pSmad3 observed in vivo. Together thesestudies show an overall decrease in the TGFb/Smad3 sig-naling pathway in macrophages in response to factors fromFGFR1-driven mammary tumor cells. The specific factorsand mechanisms responsible for this decrease are likely

Figure 6. Themigratory ability of MCF-7 cells increases with macrophagesecreted CXCR2 ligands. A, whole cell lysates were collected from thedifferent cell lines and equal amountswere used for immunoblot analysis.The expression of CXCR2 was determined and the membrane wasstained with Ponceau S for a loading control. B, THP-1 cells wereexposed to conditioned media from MCF-7 cells treated with 50 ng/mLbFGF or no treatment (NT). Cells were collected after 2 or 4 hours, andRNA was isolated for qRT-PCR for the given genes that were normalizedto cyclophilin (upper panel). For protein expression, THP-1 cells wereincubated with MCF-CM for 2 hours and then serum free media for 24hours. The conditioned media was analyzed using ELISAs (lower panel).C, MCF-CM, �/þ bFGF, was exposed to THP-1 cells for 24 hours, andthe conditioned media, MCF/THP-CM was used as the chemoattractantfor a transwell migration assay. MCF-7 cells were plated on top of theinsert with ethanol (solvent control) or CXCR2 inhibitor (þ, 100 nmol/L;þþ, 200nmol/L; andþþþ, 400nmol/L). After 18hours, themigrated cellson the bottom of the insert were stained with hematoxylin and counted.Error bars represent SEM. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.






complex and remain to be determined. Early in mammarytumor formation, TGFb is known to be growth suppressive(8), so it is possible that decreased TGFb in the microen-vironment, either from epithelial cells, infiltrating macro-phages or other stromal cells, is critical for the developmentof early stage lesions. Consistent with this, we found thatadding exogenous TGFb to the media of primary MECs in3D culture completely inhibited iFGFR1-induced acinargrowth (Supplementary Fig. 2). These results suggest that inthe early stages of tumor formation, the presence of TGFbproducing macrophages, such as those associated with thepromotion of late-stage tumors, might actually result intumor inhibition. Therefore, the association of macrophageswith decreased levels of TGFb expression and pathwayactivity, as observed in our studies, may be important forsuccessful early-stage tumor formation.Our results show that decreased expression of genes

associated with the TGFb pathway correlates with increasedexpression of inflammatory chemokines. Furthermore, res-toration of TGFb signaling with exogenous TGFb inhibitsthe expression of these chemokines. An inverse correlationbetween the TGFb pathway and expression of inflammatorychemokines has been observed in other studies. For example,decreased TGFb responsiveness in prostatic fibroblastscaused an upregulation of chemokines including CXCL1,leading to the adhesion of prostate cells to the bone matrix(31). Also, loss of TGFb signaling in mammary fibroblastsresulted in increased production of the proinflammatorychemokine Ccl2, which contributed to growth and metas-tasis of 4T1 mammary tumors (32). In addition, recentstudies using a mouse model of pancreatic ductal adenocar-cinoma also showed an increase in Cxcr2 ligands associatedwith loss of Tgfbr2 in the pancreas (24). Interestingly, thesestudies showed that induction of the Cxcr2 ligands wasdependent on the NFkB pathway, whereas induction inmacrophages in our model is dependent upon the ERKsignaling pathway. These results possibly represent cell-typespecific differences in CXCR2 ligand regulation. Takentogether, our results, along with published studies, showthat decreased TGFb signaling is associated with increasedexpression of inflammatory chemokines, and that thesechemokines are capable of contributing to tumor formationand progression.Interactions between chemokines and chemokine recep-

tors are known to contribute to breast cancer progression(33). Recent studies have implicated CXCR2 and itsvarious ligands in breast cancer. A number of studies havefocused on CXCL8, also known as IL-8, which binds toboth CXCR1 and CXCR2 and appears to be the primaryCXCR2 binding chemokine induced in response tobFGF-treated breast cancer cells (Fig. 6B). Expression ofCXCL8 has been linked to increased tumor grade andexperimental studies have showed its ability to regulateangiogenesis and tumor progression (33–35). CXCR2expression itself has been detected on breast cancer cells(34) and inhibition of CXCR2 decreased mammary tumorcell invasion in vitro (36). Furthermore, knockdown ofCXCR2 in metastatic mammary tumor cells led to

decreased metastasis in orthotopic transplantation models(36). The CXCR2 ligand CXCL3 was identified in ascreen for genes associated with basal-like breast cancersand a CXCR2 inhibitor was shown to decrease viability ofbasal-like breast cancer cell lines in vitro (37). Further-more, polymorphisms in the CXCR2 gene have beenidentified in breast cancer patients and these polymorph-isms correlated with larger tumor size, higher tumor grade,and increased lymph node metastasis (38). However, thefunctional consequences of this polymorphism onCXCR2 expression and/or activity remain to be deter-mined. Our studies show that activation of epithelial cell-specific CXCR2 by chemokine-producing cells within thetumor stroma may play a role in early stages of tumor-igenesis. Taken together, these studies suggest that theCXCR2 axis may represent a viable pathway to targetduring both early and late stage tumor development.Our studies have focused on the use of mammary epi-

thelial cells expressing an iFGFR1 construct. To show thatthe results were not specific to the iFGFR1 model, wevalidated our results using the MCF-7 cell line, which hasbeen previously used to study endogenous FGF signaling(26, 39, 40). Similar to the results obtainedwith the iFGFR1model, activation of the FGF signaling pathway in MCF-7cells also led to an increase in CXCR2 ligand gene expressionin macrophages. Interestingly, FGFR1 activity has beenshown to promote resistance of estrogen receptor positivebreast cancer cells to endocrine-based therapies (13).Although it has been reported that estrogen receptor positivecells, includingMCF-7, express lower levels of CXCR2 thanmore invasive breast cancer cell lines (34), our studies as wellas another report (41) suggest that estrogen receptor positivecells express the same or even more CXCR2 than moreinvasive breast cancer cell lines. Furthermore, our studiesshow that MCF-7 cells are capable of responding to CXCR2ligands in chemotaxis assays and that inhibition of CXCR2 issufficient to inhibit migration towards the stimulated THP-1 cells. These results suggest that activation of the FGFpathway may contribute to enhancement of tumor progres-sion by regulating the expression of tumor-promotingchemokines in infiltrating immune and other stromal cells.In addition, it is possible that FGF signaling in tumor cellsmay contribute to breast cancer growth and therapeuticresistance by regulating both the tumor cells and themicroenvironment.Although some studies have showed that breast cancer

cells produce CXCR2 ligands, which feed back to regulatetumor cell activity in an autocrine manner, our studies showthat cells within the stroma might also represent an impor-tant source of CXCR2 ligands. Similarly, studies byHalpernand colleagues showed thatmesenchymal stem cells are also asource of CXCR2 ligands, which are capable of promotingmigration of cells in vitro and suggest that CXCR2 ligandsmay be important for homing of tumor cells to bone (42).Our results show that the production of CXCR2 ligands isenhanced in macrophages in response to soluble factorsreleased from transformed mammary epithelial cells andthat these factors are then capable of promoting migration






and invasion of noninvasive cells. These results offer a novelmechanism by which macrophages associated with theinvasive edges of developing tumors might promote invasionthrough the basement membrane. Together, these resultssuggest that targeting CXCR2 in breast cancer patients maybe effective at both early and late stages of tumor formationand progression.In summary, our studies have focused on the effects of

tumor cell-specific FGFR1 activation on alterations withinthe tumor microenvironment. We have previously showeda rapid inflammatory response characterized by recruit-ment of macrophages to the epithelial structures (16). Wehave also showed that anti-inflammatory drugs are capableof inhibiting the initiation of FGFR1-driven epitheliallesions, showing that inflammation is an important pro-moter of FGFR1-driven tumorigenesis (17). Using theiFGFR1 model, we have developed novel coculture mod-els to identify specifically how FGFR1-driven tumor cellscommunicate with the microenvironment. We have iden-tified a paracrine mechanism in which soluble factors fromthe FGFR1-activated cells lead to increased production ofCXCR2 ligands, which then feed back to promote migra-tion and invasion of the tumor cells. Importantly, thesefindings were validated using an FGF-responsive breastcancer cell line, showing that activation of endogenousFGF signaling leads to similar results as the iFGFR1model. These results suggest that in FGF-driven breastcancers, targeting CXCR2 activity, possibly in conjunc-tion with specifically targeting FGFR activity, may lead toan effective therapeutic strategy. Further studies are

required to determine which patients might benefit fromthis type of therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interests were disclosed.

Authors' ContributionsConception and design: L.R. Bohrer, K.L. SchwertfegerDevelopment of methodology: L.R. Bohrer, K.L. SchwertfegerAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): L.R. Bohrer, K.L. SchwertfegerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): L.R. Bohrer, K.L. SchwertfegerWriting, review, and/or revision of the manuscript: L.R. Bohrer, K.L. SchwertfegerAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases):Study supervision: K.L. SchwertfegerPerformed the cell culture 2D and 3D coculture assays: L.R. BohrerPerformed the in vivo macrophage isolation and expression studied: K.L.Schwertfeger

AcknowledgmentsWe would like to thank Dr. Jeff Rosen for providing reagents and advice for these

studies and Dr. Fariba Behbod for providing reagents used in this study. Also, wewould like to thank Johanna Reed and Lindsey Bade for critical reading of themanuscript. In addition, we would like to acknowledge the use of the confocalmicroscope at the Masonic Cancer Center made available through an NCRR SharedInstrumentation Grant (#1 S10 RR16851).

Grant SupportFunding for these studies was provided by grants from the AmericanCancer Society

(RSG-09-192-01-LIB) and NCI (1R01CA132827) to KLS.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received May 3, 2012; revised July 5, 2012; accepted July 18, 2012;published OnlineFirst August 14, 2012.

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Published OnlineFirst August 14, 2012.Mol Cancer Res Laura R. Bohrer and Kathryn L. Schwertfeger Cxcr2-Dependent MannerReceptor-Driven Tumor Cell Migration and Invasion in a Macrophages Promote Fibroblast Growth Factor

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